RESUMO
Peroxidases have received considerable attention as a cost-effective and environmentally friendly catalyst for bioremediation. Their rapid activity loss under harsh environmental conditions and inability to be used repetitively limit their exploitation in real-world wastewater treatment. First, a peroxidase was produced extracellularly by Bacillus mojavensis TH309 and purified 8.12-fold with a final yield of 47.10â¯% using Sephadex G-100 superfine resin. The pure peroxidase (BmPer) possessed a relatively low molecular weight of ~21â¯kDa and was active against L-DOPA on acrylamide gel after electrophoresis. BmPer was immobilized by adsorption functionalized walnut shell hydrochar (WsH) with 61.99⯱â¯1.34â¯% efficiency and 37.07⯱â¯4.16â¯% activity loss. BmPer and its immobilized form (WsH-BmPer) exhibited maximum activity at 50⯰C and pHâ¯9. WsH-BmPer exhibited 3.23-, 2.37-, 1.65-, and 2.25-fold longer half-life than BmPer at 50, 60, 70, and 80⯰C, respectively. Immobilization significantly enhanced the stability of the enzyme under acidic conditions. BmPer and WsH-BmPer showed maximal activity in the presence of 1â¯% salt and retained more than 85â¯% of their activity even after pre-incubation with 2.5â¯M salt for 60â¯min at 50⯰C. Their catalytic efficiency was significantly stimulated by pre-incubation with Triton X-100 (1â¯mM), Tween20 (1â¯mM), and Mg2+ (1 and 10â¯mM). Immobilization strongly reduced the loss of activity caused by inhibitors including Ba2+, Hg2+, and Cu2+. Moreover, both forms of the enzyme were compatible with solvents. The Michaelis constant (Km) values of BmPer and WsH-BmPer were 0.88 and 2.66â¯mM for 2,4 DCP, respectively. WsH-BmPer peroxidase maintained about 82â¯% and 85â¯% of its activity when stored at 4⯰C for 30â¯days and reused for up to 10â¯cycles, respectively. Furthermore, it decolorized Cibacron red (CR), Poly R-478 (PR), Remazol Brilliant Blue R (RBBR), and Methyl red (MR) dyes by 60.13â¯%, 91.34â¯%, 86.41â¯%, and 50.51â¯% within 60â¯min, respectively.
RESUMO
Laccases or laccase-like multicopper oxidases have great potential in bioremediation to oxidase phenolic or non-phenolic substrates. However, their inability to maintain stability in harsh environmental conditions and against non-substrate compounds is one of the main reasons for their limited use. The gene (mco) encoding multicopper oxidase from Bacillus mojavensis TH309 were cloned into pET14b( +), expressed in Escherichia coli, and purified as histidine tagged enzyme (BmLMCO). The molecular weight of the enzyme was about 60 kDa. The enzyme exhibited laccase-like activity toward 2,6-dimethoxyphenol (2,6-DMP), syringaldazine (SGZ), and 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS). The highest enzyme activity was recorded at 80 °C and pH 8. BmLMCO showed a half-life of ~ 305, 99, 50, 46, 36, and 20 min at 40, 50, 60, 70, 80, and 90 °C, respectively. It retained more than 60% of its activity after pre-incubation in the range of pH 5-12 for 60 min. The enzyme activity significantly increased in the presence of 1 mM of Cu2+. Moreover, BmLMCO tolerated various chemicals and showed excellent compatibility with organic solvents. The Michaelis constant (Km) and the maximum velocity (Vmax) values of BmLMCO were 0.98 mM and 93.45 µmol/min, respectively, with 2,6-DMP as the substrate. BmLMCO reduced the antibacterial activity of cefprozil, gentamycin, and erythromycin by 72.3 ± 1.5%, 79.6 ± 6.4%, and 19.7 ± 4.1%, respectively. This is the first revealing shows the recombinant production of laccase-like multicopper oxidase from any B. mojavensis strains, its biochemical properties, and potential for use in bioremediation.